Vitamin C, or ascorbic acid, has been shown in animal and clinical studies to help delay or prevent complications attributed to increased oxidant stress in stroke and in several neurodegenerative disorders. Normally, ascorbate is maintained at relatively high concentrations in brain and especially in neurons, where it serves as an antioxidant and supports several important neuronal functions. Ascorbate concentrations in the low millimolar range are generated in neurons by a specific ascorbate transporter, termed the SVCT2. Mice engineered to lack this transporter have very low brain ascorbate contents and die at birth with cerebral hemorrhage, indicating that the vitamin is crucial for survival. The overall goal of this proposal is to determine how the SVCT2 transporter is regulated in neurons, and whether changes in transporter number can affect neuronal function and antioxidant defenses, both in vitro and in vivo. There are three specific aims. In the first aim, mechanisms of SVCT2 regulation and structure-function relationships will be evaluated in cultured SH-SY5Y neuroblastoma cells and in Xenopus oocytes. Regulation of both SVCT2 expression and activity in response to oxidant stress will be assessed in SH-SY5Y cells. Further, the functional role of key cysteines on the extracellular surface of the protein will be evaluated by affinity labeling in SH- SY5Y cells and by mutagenesis approaches in Xenopus oocytes. In the second aim, the mechanism by which knock-out mice lacking the SVCT2 die at birth will be determined. Also in Aim 2, a novel model of oxidant stress due to combined deficiencies of vitamins E and C will be used to establish whether transgenic mice expressing increased amounts of SVCT2 are protected from neurologic damage compared to mice expressing normal or decreased (50%) SVCT2. In the third aim, primary culture neurons will be prepared from mice expressing several different levels of SVCT2 (transgenic, wild-type, heterozygous knock-out, and homozygous knock-out). These cells will be used to test whether SVCT2 expression 1) determines the ability of neurons to maintain intracellular ascorbate, 2) increases resistance of neurons to oxidant stress, 3) enhances maturation of cortical neurons in culture, and 3) affects neuronal electrophysiology. Results from these studies are expected to show that the SVCT2 ascorbate transporter, as regulated by oxidant stress, is crucial for neuronal function, development, and survival. PUBLIC HEALTH RELEVANCE: This project to study the function, regulation and tissue expression of the transporter for vitamin C in the brain has relevance for neuronal function, maturation and resistance to oxidant stress. Several neurodegenerative diseases including Alzheimer's and Parkinson's are associated with increased oxidant stress so that this project will help define the role of vitamin C in oxidant injury to the brain.